Development of prophylactic and therapeutic vaccines targeting infectious diseases and cancer is of prime importance for the health system and the rest of the world. The discovery of powerful adjuvant is also of paramount interest, as they play a key role in the modulation of an effective immune response toward antigens of interest used in vaccination. While studying the importance of hemozoin (HZ) in the modulation of a host inflammation, it was discovered that this inert crystalline pigment and its synthetic counterpart, hematin anhydride (HA), were effectively strongly recognized by the innate immune system.
Malaria pigment, also termed HZ, is comprised of black prismatic crystals of heme produced by the Plasmodium parasite upon hemoglobin (Hb) catabolism. HZ chemical structure renders it difficult to degrade making it long lasting in the body, property highly requested in vaccine development. Recently, it was demonstrated that HZ and/or HA can activate the NLRP3-inflammasome resulting in the production of IL-1β by macrophages, similarly to alum. It was further shown that, when administered with soluble Leishmania antigen, animals are effectively protected against infectious challenge. Others have also demonstrated the adjuvant properties of HZ when co-administered with OVA, HVA and house dust mite allergen in mice, beagle dogs and non-human primates. Taken together the data supports the potential development of HA as an adjuvant, and also suggests that HA favors a Th1 type cellular immune response.
Various synthesis protocols have been elaborated for HA, such as seeding with HZ, catalysis by remnant parasite biomolecules, catalysis by lipids, alcohol or chloroform-water interface, acidic precipitation from alkaline solution of hemin or hematin, and anhydrous base-annealing method. However, none of these methods yield exact biomimetics of natural HZ in term of size and crystal morphology.
Much efforts have been put towards the development an accessible synthesis protocol of HA. International patent application publication WO2007147255 describes a protocol for using HA as an adjuvant where antigens are coated on the surface of HA crystals. HA crystals are being synthesized using an acid-catalyzed precipitation because it uses simple bench-top reactions with accessible chemicals, which is very convenient for researchers in the medical and biochemistry field. Previous protocols available in the literature exploited this acidic precipitation from alkali solution. Unfortunately non-reproducible and mostly aggregated material of poor crystallinity was obtained using these methods. Although this process is rapid and simple, homogenous and reproducible heme crystalline phase of HZ size can result when some parameters are carefully controlled. However, even after best optimization, this method yields aggregates of microcrystalline domains, very different from the single domain parallelepipeds of Plasmodium HZ.